Wendy S. Hill

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C‐terminal 343 amino acids of GAP (residues 702‐1044) were observed to bind Ha ras p21‐GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M‐1 s‐1 at 24 degrees C) as GAP purified from bovine brain or full‐length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986‐1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C‐II, the crk oncogene product, and the non‐receptor tyrosine kinase oncogene products. This homologous region is located in the N‐terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

Identification of Residues Essential for the Active Conformation of the ß-Adrenergic Receptor by Site-Directed Mutagenesis

Many hormones and neurotransmitters mediate their actions by stimulating one of a class of receptors which are functionally coupled to guanine nucleotide binding regulatory proteins (G-proteins). Activation of the G-protein by the agonist-occupied form of the receptor causes the release of GDP from the G-protein, allowing GTP to bind. The GTP-bound form of the G-protein is the active form, which dissociates from the receptor, leading to the activation or inhibition of specific effector enzymes and modulating levels of intracellular second messengers. The elucidation of the primary sequences of several G-protein coupled receptors by molecular cloning has shown them to share characteristic structural elements, presumably related to their common mechanism of action (see Dixon, et al, 1989, for review). All of these receptors whose sequences are known contain 7 stretches of conserved hydrophobic amino acids, thought to form transmembrane α- helices, which connect 8 more divergent hydrophilic regions, postulated to form alternating extracellular and intracellular loops. In our laboratory, we have used genetic anaiysis of-the s-adrenergic receptor (sAR), which is linked to GS to stimulate adenylyl cyclase, to identify structural domains and specific amino acid residues required for receptor function. A model for the structure of the sAR is shown in Figure 1. From these studies and from biochemical and genetic analysis of other G-protein coupled receptors, a model for receptor activity is emerging, which may serve to identify potential sites of pharmacological intervention within these signal transduction systems.

The ras Oncogene Protein

To date, over 40 discrete genes (oncogenes and proto-oncogenes) have been shown capable of inducing cellular transformation and tumor development (Bishop 1987). Only a few of these oncogenes have actually been shown to have a significant role in human cancer. The proto-oncogenes bc1-2, c-myc, c-abl, c-ets, and c-myb are altered by chromosomal translocations in specific tumors. Amplification of the c-erbB, c-myc, L-myc, and N-myc proto-oncogenes occurs in a high frequency of other tumor types. Transfection of NIH3T3 cells with DNA isolated from human tumors frequently identifies the Ha-ras, Ki-ras, N-ras, met, mel, trk, dbl, and c-raf-1 oncogenes (Martin-Zanca et al. 1986; Ron et al. 1988).

Molecular Biology of GAP and its Interaction with Oncogenic ras p21

The interaction of ras p21 with guanine nucleotides is central to its biological function. Mutations that impair the intrinsic GTPase or that enhance GTP for GDP exchange, activate ras cell-transforming activity. Both of these types of mutations result in an increased level of ras p21-GTP complex. By analogy with G-protein regulatory cycles therefore, the ras p21-GTP complex would be predicted to be the biologically active species. This prediction appears to be true for both the mammalian and yeast systems. Discrepancies between the in vitro GTPase activities and biological potencies which were observed, prompted the testing of the model through the quantitation of the GTP and GDP nucleotides bound to both normal and oncogenic ras proteins in vivo. These measurements were first made for ras proteins expressed in yeast cells where the ras proteins could be overproduced. Yeast RAS1 and RAS2 proteins, overexpressed in [32P] orthophosphate-labelled cultures of S. cerevisiae cells, were found to be bound almost entirely to GDP, whereas increased amounts of GTP were bound to RAS proteins containing oncogenic mutations that impair GTPase activity. Unexpectedly, normal mammalian ras p21 was bound to near equimolar proportions of both GTP and GDP nucleotides, whereas the oncogenic forms were bound almost exclusively to GTP (Gibbs et al., 1987).